Microorganisms and methods for producing pyruvate, ethanol, and other compounds

ABSTRACT

Microorganisms comprising modifications for producing pyruvate, ethanol, and other compounds. The microorganisms comprise modifications that reduce or ablate activity of one or more of pyruvate dehydrogenase, 2-oxoglutarate dehydrogenase, phosphate acetyltransferase, acetate kinase, pyruvate oxidase, lactate dehydrogenase, cytochrome terminal oxidase, succinate dehydrogenase, 6-phosphogluconate dehydrogenase, glutamate dehydrogenase, pyruvate formate lyase, pyruvate formate lyase activating enzyme, and isocitrate lyase. The microorganisms optionally comprise modifications that enhance expression or activity of pyruvate decarboxylase and alcohol dehydrogenase. The microorganisms are optionally evolved in defined media to enhance specific production of one or more compounds. Methods of producing compounds with the microorganisms are provided.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under DE-FC02-07ER64494,DE-SC0008103 awarded by the US Department of Energy. The government hascertain rights in the invention.

BACKGROUND

Over the past decade a number of chemical companies have begun todevelop infrastructures for the production of compounds using bio-basedprocesses. Considerable progress has been reported toward new processesfor producing commodity chemicals such as ethanol, lactic acid,1,3-propanediol, and adipic acid. In addition, advances have been madein the genetic engineering of microbes for higher value specialtycompounds such as acetate, polyketides, and carotenoids.

Pyruvate is a starting material for synthesizing a variety of biofuelsand chemicals. Industrially, pyruvate is produced via dehydration anddecarboxylation of calcium tartrate, a byproduct of the wine industry.This process involves toxic solvents and is energy intensive with anestimated production cost of $8,650 per ton of pyruvate. Microbialpyruvate production is based primarily upon two microorganisms, amulti-vitamin auxotroph of the yeast T. glabrata and a lipoic auxotrophof E. coli containing an F1ATPase mutation. The estimated cost ofpyruvate production via microbial fermentation with such strains isestimated to be $1,255 per ton of pyruvate, an 85% savings. Increasingthe yield of pyruvate would increase the savings even further.

Ethanol is mainly of interest as a petrol additive, or substitute,because ethanol-blended fuel produces a cleaner, more completecombustion that reduces greenhouse gas and toxic emissions. Theproduction of ethanol in the US has increased tremendously in recentyears, and demand is projected to increase even further. As aconsequence of the surge in demand for biofuels, ethanol-producingmicroorganisms are of considerable interest due to their potential forthe production of bioethanol. To keep in step with the growing demandfor biofuels, the engineering of new strains of fermentativemicroorganisms that can efficiently produce ethanol will be required.

There is a need for microorganisms that efficiently produce pyruvate,ethanol, or other commodity chemicals.

SUMMARY OF THE INVENTION

The present invention addresses the aforementioned needs by providingmicroorganisms with increased production of pyruvate, ethanol, or othercommodity chemicals. Methods of producing commodity chemicals with themicroorganisms described herein are also provided.

One aspect of the invention is a microorganism comprising modificationsthat reduce or ablate activity of one or more enzymes in a first set,one or more enzymes in a second set, and enzymes in a third set. Theenzymes in the first set are selected from the group consisting ofpyruvate dehydrogenase and 2-oxoglutarate dehydrogenase. The enzymes inthe second set are selected from the group consisting of phosphateacetyltransferase, acetate kinase, and pyruvate oxidase. The enzymes inthe third set comprise lactate dehydrogenase and one or more enzymesselected from the group consisting of cytochrome terminal oxidase andsuccinate dehydrogenase; lactate dehydrogenase and one or more enzymesselected from the group consisting of 6-phosphogluconate dehydrogenaseand glutamate dehydrogenase; one or more enzymes selected from the groupconsisting of cytochrome terminal oxidase and succinate dehydrogenaseand one or more enzymes selected from the group consisting of6-phosphogluconate dehydrogenase and glutamate dehydrogenase; or lactatedehydrogenase, one or more enzymes selected from the group consisting ofcytochrome terminal oxidase and succinate dehydrogenase, and one or moreenzymes selected from the group consisting of 6-phosphogluconatedehydrogenase and glutamate dehydrogenase.

In some versions, the one or more enzymes in the first set are selectedfrom pyruvate dehydrogenase.

In some versions, the one or more enzymes in the second set are selectedfrom the group consisting of phosphate acetyltransferase and pyruvateoxidase.

In some versions, the enzymes in the third set comprise lactatedehydrogenase and cytochrome terminal oxidase, lactate dehydrogenase andone or more enzymes selected from the group consisting of6-phosphogluconate dehydrogenase and glutamate dehydrogenase, orsuccinate dehydrogenase and 6-phosphogluconate dehydrogenase.

In some versions, the one or more enzymes in the first set are selectedfrom pyruvate dehydrogenase, the one or more enzymes in the second setare selected from phosphate acetyltransferase, and the enzymes in thethird set comprise lactate dehydrogenase and one or more enzymesselected from the group consisting of cytochrome terminal oxidase andsuccinate dehydrogenase, or lactate dehydrogenase and one or moreenzymes selected from the group consisting of 6-phosphogluconatedehydrogenase and glutamate dehydrogenase.

In some versions, the one or more enzymes in the first set are selectedfrom pyruvate dehydrogenase, the one or more enzymes in the second setare selected from phosphate acetyltransferase, and the enzymes in thethird set comprise lactate dehydrogenase and cytochrome terminaloxidase, or lactate dehydrogenase and one or more enzymes selected fromthe group consisting of 6-phosphogluconate dehydrogenase and glutamatedehydrogenase.

In some versions, the one or more enzymes in the first set are selectedfrom pyruvate dehydrogenase, the one or more enzymes in the second setare selected from pyruvate oxidase, and the enzymes in the third setcomprise one or more enzymes selected from the group consisting ofcytochrome terminal oxidase and succinate dehydrogenase and one or moreenzymes selected from the group consisting of 6-phosphogluconatedehydrogenase and glutamate dehydrogenase.

In some versions, the microorganism further comprises a modificationthat reduces or ablates activity of an enzyme selected from the groupconsisting of pyruvate formate lyase and pyruvate formate lyaseactivating enzyme.

In some versions, the microorganism further comprises a modificationthat enhances expression of pyruvate decarboxylase and alcoholdehydrogenase.

In some versions, the microorganism is a bacterium or a yeast.

In some versions, an evolved microorganism is produced by sequentiallyculturing any microorganism described above or elsewhere herein in mediacomprising decreasing concentrations of a compound such as acetate,ethanol, or another compound. The media each preferably compriseapproximately a same amount of total consumable carbon. In someversions, the microorganism is cultured in media comprising decreasingconcentrations of acetate. The concentrations of acetate in the mediamay range from about 0.1 mg/L acetate to about 3 g/L acetate.

Another aspect of the invention is a method of producing a chemical. Themethod comprises culturing any microorganism described above orelsewhere herein. The chemical may be selected from the group consistingof pyruvate and ethanol. The culturing may comprise culturing themicroorganism in a medium comprising a biomass hydrolysate.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schema showing the central metabolic pathway of wild-type E.coli. Genes associated with each reaction in the central metabolicnetwork are shown and flux values are labeled. The metabolic fluxdistribution for the wild-type strain under aerobic conditions waspredicted by flux balance analysis. Glucose uptake rate was set at 10mmol/gDW/hour. The dashed line represents the ethanol synthesis pathway(PET operon) from Zymomonas mobilis.

FIGS. 2A-2D are schemas showing the central metabolic pathway of mutantE. coli strains designed for pyruvate production. Genes associated witheach reaction in the central metabolic network are shown and flux valuesare labeled. The reactions marked by bars correspond to the deletiontargets calculated computationally. The labeled metabolic fluxdistribution for each strain was predicted by flux balance analysis.Glucose uptake rate was set at 10 mmol/gDW/hour. Oxygen uptake wasunlimited for the strains shown in FIGS. 2B-2D, but limited to 3mmol/gDW/hour for the strain shown in FIG. 2A. FIG. 2A: Strain designedas ΔaceE, ΔcyoA, ΔcydB, Δpta, ΔeutI, ΔidhA, and Δdld. FIG. 2B: Straindesigned as ΔlpdA, Δgnd, ΔsdhA, ΔpoxB, ΔpflB, ΔpflD, ΔtdcE, and ΔpurU.FIG. 2C: Strain designed as ΔaceE, ΔgdhA, ΔpoxB, ΔldhA, Δdld, ΔatpE,ΔpflB, ΔpflD, and ΔtdcE. FIG. 2D: Strain designed as designed as ΔaceE,Δgnd, ΔpoxB, ΔldhA, Δdld, ΔatpE, ΔpflB, ΔpflD, and ΔtdcE.

FIGS. 3A-3F show growth (FIGS. 3A and 3D), pyruvate production (FIGS. 3Band 3E), and glucose consumption (FIGS. 3C and 3F) of wild-type(BW25113) and mutant E. coli strains. Cells were grown in M9 minimalmedium containing glucose and acetate. (See Table 2 for media details).

FIG. 4 shows (a) lactate and (b) acetate secretion for parent (BW25113)and mutant E. coli strains under aerobic conditions in shake flasks. Theshown concentrations are the maximum acid concentrations observed over60 hours during growth in M9 minimal medium supplemented with glucoseand acetate. (See Table 2 for media details). Acetate accumulated inBW25113, PYR001 and PYR002 cultures and lactate accumulated in PYR002cultures. * indicates concentrations of acetate and lactate that werebelow the detection level of the HPLC.

FIG. 5 shows growth, glucose consumption, and pyruvate production byPYR004 in bioreactors. Panels (A) and (B) show batch fermentation inminimal salts medium containing 30 g/L glucose with 1.5 g/L acetate(panel A) or 3 g/L acetate (panel B). Panel (C) shows fed-batchfermentation operated in minimal salts medium initially containing 30g/L glucose and 1.5 g/L acetate. In the fed-batch operation, anadditional 7.5 mL of 200 g/L acetate was added at 8.5 hours, indicatedby the black arrow, for a total acetate concentration of 3.0 g/L.Experiments were performed in duplicate. Diamond: OD 600. Triangle:glucose concentration. Square: pyruvate concentration.

FIG. 6 shows growth, glucose consumption and pyruvate production byPYR020 in bioreactors. Panels (A) and (B) show batch fermentation inminimal salts medium containing 30 g/L glucose with 0.9 g/L acetate(Panel A) or 1.5 g/L acetate (Panel B). Panel (C) shows fed-batchfermentation operated in minimal salts medium initially containing 30g/L glucose and 0.6 g/L acetate. In the fed-batch operation, anadditional 1.5 mL of 200 g/L acetate was added at 17 hours, indicated bythe black arrow. Experiments were performed in duplicate. Diamond: OD600. Triangle: glucose concentration. Square: pyruvate concentration.

FIG. 7 shows batch production of pyruvate in ammonia fiber expansion(AFEX)-pretreated switchgrass hydrolysate (ASGH) by strain PYR020. Cellswere grown in ASGH containing 48 g/L glucose, 27 g/L xylose, and 2.6 g/Lacetate. Diamond: OD 600. Square: pyruvate concentration.

FIGS. 8A-8B show product secretion from various strains under anaerobicconditions. Secretion of ethanol, succinate, and formate is shown inFIG. 8A. Secretion of acetate and lactate is shown in FIG. 8B. Allexperiments were performed anaerobically in hungate tubes in M9 minimalmedia. Columns marked “a” correspond to fermentations containing 1.98g/L glucose and 0.02 g/L acetate. Multiple samples were taken over 48hours, which reduced the culture volume by about 50%. Columns marked (b)correspond to fermentations in M9 medium with 1.98 g/L glucose and 0.02g/L acetate for 24 hours, but only three samples were taken at 16, 20and 24 hours. Columns marked (c) correspond to fermentations in M9minimal medium with more acetate (0.1 g/L) and 1.9 g/L glucose for 24hours, with only three samples. Error bars represent standard errorsamong three replicates. Percent of theoretical yield was calculated asthe ethanol concentration divided by the theoretical maximum productionof ethanol (2 mmol of ethanol per mmol of glucose plus 0.67 mmol ofethanol per mmol of acetate). t-tests were used to determine significantdifferences in product concentrations between different fermentations(a, b, and c columns) where * and ** indicates the p-value is between0.01 and 0.05, or less than 0.01, respectively.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention is directed to microorganisms comprisingmodifications that reduce or ablate the activity of gene products of oneor more genes. Such a modification that that reduces or ablates theactivity of gene products of one or more genes is referred to herein asa “functional deletion” of the gene product. “Gene product” refers to aprotein or polypeptide encoded and produced by a particular gene. “Gene”refers to a nucleic acid sequence capable of producing a gene productand may include such genetic elements as a coding sequence together withany other genetic elements required for transcription and/or translationof the coding sequence. Such genetic elements may include a promoter, anenhancer, and/or a ribosome binding site (RBS), among others.

One of ordinary skill in the art will appreciate that there are manywell-known ways to functionally delete a gene product. For example,functional deletion can be accomplished by introducing one or moregenetic modifications. As used herein, “genetic modifications” refer toany differences in the nucleic acid composition of a cell, whether inthe cell's native chromosome or in endogenous or exogenousnon-chromosomal plasmids harbored within the cell. Examples of geneticmodifications that may result in a functionally deleted gene productinclude but are not limited to mutations such as substitutions, partialor complete deletions, insertions, or other variations to a codingsequence or a sequence controlling the transcription or translation of acoding sequence; placing a coding sequence under the control of a lessactive promoter; blocking transcription of the gene with a trans-actingDNA binding protein such as a TAL effector or CRISPR guided Cas9; andexpressing ribozymes or antisense sequences that target the mRNA of thegene of interest, etc. In some versions, a gene or coding sequence canbe replaced with a selection marker or screenable marker. Variousmethods for introducing the genetic modifications described above arewell known in the art and include homologous recombination, among othermechanisms. See, e.g., Green et al., Molecular Cloning: A laboratorymanual, 4^(th) ed., Cold Spring Harbor Laboratory Press (2012) andSambrook et al., Molecular Cloning: A Laboratory Manual, 3^(rd) ed.,Cold Spring Harbor Laboratory Press (2001). Various other geneticmodifications that functionally delete a gene product are described inthe examples below. Functional deletion can also be accomplished byinhibiting the activity of the gene product, for example, by chemicallyinhibiting a gene product with a small molecule inhibitor, by expressinga protein that interferes with the activity of the gene product, or byother means.

In certain versions of the invention, the functionally deleted geneproduct may have less than about 95%, less than about 90%, less thanabout 85%, less than about 80%, less than about 75%, less than about70%, less than about 65%, less than about 60%, less than about 55%, lessthan about 50%, less than about 45%, less than about 40%, less thanabout 35%, less than about 30%, less than about 25%, less than about20%, less than about 15%, less than about 10%, less than about 5%, lessthan about 1%, or about 0% of the activity of the non-functionallydeleted gene product.

In certain versions of the invention, a cell with a functionally deletedgene product may have less than about 95%, less than about 90%, lessthan about 85%, less than about 80%, less than about 75%, less thanabout 70%, less than about 65%, less than about 60%, less than about55%, less than about 50%, less than about 45%, less than about 40%, lessthan about 35%, less than about 30%, less than about 25%, less thanabout 20%, less than about 15%, less than about 10%, less than about 5%,less than about 1%, or about 0% of the activity of the gene productcompared to a cell with the non-functionally deleted gene product.

In certain versions of the invention, the functionally deleted geneproduct may be expressed at an amount less than about 95%, less thanabout 90%, less than about 85%, less than about 80%, less than about75%, less than about 70%, less than about 65%, less than about 60%, lessthan about 55%, less than about 50%, less than about 45%, less thanabout 40%, less than about 35%, less than about 30%, less than about25%, less than about 20%, less than about 15%, less than about 10%, lessthan about 5%, less than about 1%, or about 0% of the amount of thenon-functionally deleted gene product.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least 1, atleast 2, at least 3, at least 4, at least 5, at least 10, at least 20,at least 30, at least 40, at least 50, or more nonsynonymoussubstitutions are present in the gene or coding sequence of the geneproduct.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least 1, atleast 2, at least 3, at least 4, at least 5, at least 10, at least 20,at least 30, at least 40, at least 50, or more bases are inserted in thegene or coding sequence of the gene product.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least about1%, at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100% of the gene product's gene or coding sequenceis deleted or mutated.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least about1%, at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100% of a promoter driving expression of the geneproduct is deleted or mutated.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least about1%, at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100% of an enhancer controlling transcription of thegene product's gene is deleted or mutated.

In certain versions of the invention, the functionally deleted geneproduct may result from a genetic modification in which at least about1%, at least about 5%, at least about 10%, at least about 15%, at leastabout 20%, at least about 25%, at least about 30%, at least about 35%,at least about 40%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, at least about 75%,at least about 80%, at least about 85%, at least about 90%, at leastabout 95%, or about 100% of a sequence controlling translation of geneproduct's mRNA is deleted or mutated.

In certain versions of the invention, the decreased activity orexpression of the functionally deleted gene product is determined withrespect to the activity or expression of the gene product in itsunaltered state as found in nature. In certain versions of theinvention, the decreased activity or expression of the functionallydeleted gene product is determined with respect to the activity orexpression of the gene product in its form in a correspondingmicroorganism. In certain versions, the genetic modifications givingrise to a functionally deleted gene product are determined with respectto the gene or coding sequence in its unaltered state as found innature. In certain versions, the genetic modifications giving rise to afunctionally deleted gene product are determined with respect to thegene or coding sequence in its form in a corresponding microorganism.

As used herein, “corresponding microorganism” refers to a microorganismof the same species having the same or substantially same genetic andproteomic composition as a microorganism of the invention, with theexception of genetic and proteomic differences resulting from themodifications described herein for the microorganisms of the invention.

Some versions of the invention comprise microorganisms configured forincreased production of pyruvate. For the production of pyruvate, atleast three sets of enzymes are functionally deleted in themicroorganism. Enzymes in a first set are selected from the groupconsisting of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase.Enzymes in a second set are selected from the group consisting ofphosphate acetyltransferase, acetate kinase, and pyruvate oxidase.Enzymes in a third set comprise lactate dehydrogenase and one or moreenzymes selected from the group consisting of cytochrome terminaloxidase and succinate dehydrogenase; lactate dehydrogenase and one ormore enzymes selected from the group consisting of 6-phosphogluconatedehydrogenase and glutamate dehydrogenase; one or more enzymes selectedfrom the group consisting of cytochrome terminal oxidase and succinatedehydrogenase and one or more enzymes selected from the group consistingof 6-phosphogluconate dehydrogenase and glutamate dehydrogenase; orlactate dehydrogenase, one or more enzymes selected from the groupconsisting of cytochrome terminal oxidase and succinate dehydrogenase,and one or more enzymes selected from the group consisting of6-phosphogluconate dehydrogenase and glutamate dehydrogenase. Deletionof any gene or any other modification that reduces or ablates theactivity of these enzymes or reduces or ablates flux of metabolitesthrough these enzymes is encompassed by the present invention.

Pyruvate dehydrogenases convert pyruvate into acetyl Co-A. Pyruvatedehydrogenases include enzymes classified under any or all of EC1.2.4.1, EC 2.3.1.12, and EC 1.8.1.4. An exemplary pyruvatedehydrogenase is the pyruvate dehydrogenase of E. coli, which is amulti-subunit complex comprising AceE (SEQ ID NO:2) encoded by aceE (SEQID NO:1), AceF (SEQ ID NO:4) encoded by aceF (SEQ ID NO:3), and Lpd (SEQID NO:6) encoded by lpdA (SEQ ID NO:5). AceE has activity classifiedunder EC 1.2.4.1. AceF has activity classified under 2.3.1.12. Lpd hasactivity classified under 1.8.1.4. Other pyruvate dehydrogenases includehomologs of the E. coli pyruvate dehydrogenase.

2-Oxoglutarate dehydrogenases convert α-ketoglutarate, NAD⁺, and CoA tosuccinyl CoA, CO₂, and NADH. 2-Oxoglutarate dehydrogenases includeenzymes classified under any one or all of EC 1.8.1.4, EC 1.2.4.2, andEC 2.3.1.61. An exemplary 2-oxoglutarate dehydrogenase is the2-oxoglutarate dehydrogenase of E. coli, which is a multi-subunitcomplex comprising Lpd (SEQ ID NO:6) encoded by lpdA (SEQ ID NO:5), SucA(SEQ ID NO:8) encoded by sucA (SEQ ID NO:7), and SucB (SEQ ID NO:10)encoded by sucB (SEQ ID NO:9). Lpd has activity classified under EC1.8.1.4. SucA has activity classified under EC 1.2.4.2. SucB hasactivity classified under EC 2.3.1.61. Other 2-oxoglutaratedehydrogenases include homologs of the E. coli 2-oxoglutaratedehydrogenase. Functionally deleting 2-oxoglutarate dehydrogenase may beperformed as an alternative to or in addition to functionally deletingpyruvate dehydrogenase.

Phosphate acetyltransferases convert acetyl-CoA and phosphate to CoA andacetyl phosphate. Phosphate acetyltransferases include enzymesclassified under EC 2.3.1.8. An exemplary phosphate acetyltransferase isthe phosphate acetyltransferase of E. coli (SEQ ID NO:12), which isencoded by pta (SEQ ID NO:11). Other phosphate acetyltransferasesinclude homologs of the E. coli phosphate acetyltransferase.

Acetate kinases convert acetate and ATP to acetyl phosphate. Acetatekinases include enzymes classified under EC 2.7.2.-, such as EC 2.7.2.1.An exemplary acetate kinase is the acetate kinase A of E. coli (SEQ IDNO:14), which is encoded by ackA (SEQ ID NO:13). Other acetate kinasesinclude homologs of the E. coli acetate kinase A. Functionally deletingacetate kinase may be performed as an alternative to or in addition tofunctionally deleting phosphate acetyltransferase. In some versions, theackA gene in the microorganism is structurally and functionally intactsuch that the acetate kinase in the cells is fully expressed and fullyfunctional.

Pyruvate oxidases convert pyruvate, phosphate, and O₂ to acetylphosphate, CO₂, and H₂O₂. Pyruvate oxidases include enzymes classifiedunder EC 1.2.3.3. An exemplary pyruvate oxidase is the pyruvate oxidaseof E. coli (SEQ ID NO:16), which is encoded by poxB (SEQ ID NO:15).Other pyruvate oxidases include homologs of the E. coli pyruvateoxidase.

Lactate dehydrogenases convert pyruvate to lactate and vice versa.Lactate dehydrogenases include enzymes classified under any or all of EC1.1.1.27 and EC 1.1.1.28. An exemplary lactate dehydrogenase is the LdhAof E. coli (SEQ ID NO:18), which is encoded by ldhA (SEQ ID NO:17).Other lactate dehydrogenases include homologs of the E. coli LdhA.

Cytochrome oxidases transfer electrons in the respiratory chain fromdonors to an acceptor. Cytochrome oxidases include enzymes classifiedunder any or all of EC 1.9.3.1 and EC 1.10.3.-. Exemplary cytochromeoxidases suitable for functionally deleting in the present inventioninclude cytochrome terminal oxidases, such as Family A cytochrometerminal oxidases. An exemplary Family A cytochrome terminal oxidase inE. coli is the cytochrome bo terminal oxidase, which is a multi-subunitcomplex comprising subunit I (SEQ ID NO:22) encoded by cyoB (SEQ IDNO:21), subunit II (SEQ ID NO:20) encoded by cyoA (SEQ ID NO:19),subunit III (SEQ ID NO:24) encoded by cyoC (SEQ ID NO:23), and subunitIV (SEQ ID NO:26) encoded by cyoD (SEQ ID NO:25). Subunits I-IV haveactivity classified under EC 1.10.3.-. A fifth gene of the cyo operon,cyoE (SEQ ID NO:27), encodes a heme 0 synthase (SEQ ID NO:28) that isessential for correct assembly of the complex and can be functionallydeleted to effectively functionally delete the cytochrome bo terminaloxidase itself. Other cytochrome oxidases include homologs of the E.coli cytochrome bo terminal oxidase.

Succinate dehydrogenases catalyze the oxidation of succinate to fumaratewith the reduction of ubiquinone to ubiquinol. Succinate dehydrogenasesinclude enzymes classified under EC 1.3.5.1. An exemplary succinatedehydrogenase is the succinate dehydrogenase of E. coli, which is amulti-subunit complex comprising SdhA (SEQ ID NO:30) encoded by sdhA(SEQ ID NO:29), SdhB (SEQ ID NO:32) encoded by sdhB (SEQ ID NO:31), SdhC(SEQ ID NO:34) encoded by sdhC (SEQ ID NO:33), and SdhD (SEQ ID NO:36)encoded by sdhD (SEQ ID NO:35). Other succinate dehydrogenases includehomologs of the E. coli succinate dehydrogenases.

6-Phosphogluconate dehydrogenases catalyze the decarboxylating reductionof 6-phosphogluconate into ribulose 5-phosphate in the presence ofNADP⁺. Phosphogluconate dehydrogenases include enzymes classified underEC 1.1.1.44. An exemplary 6-phosphogluconate dehydrogenase is the Gnd ofE. coli (SEQ ID NO:38), which is encoded by gnd (SEQ ID NO:37). Other6-phosphogluconate dehydrogenases include homologs of the E. coli Gnd.

Glutamate dehydrogenases convert glutamate to α-ketoglutarate and viceversa. Glutamate dehydrogenases include enzymes classified under EC1.4.1.4. An exemplary glutamate dehydrogenase is the GdhA of E. coli(SEQ ID NO:40), which is encoded by gdhA (SEQ ID NO:39). Other glutamatedehydrogenases include homologs of the E. coli GdhA.

In some versions of the invention, the microorganisms having theabove-referenced sets of enzymes functionally deleted are evolved forenhanced production of pyruvate. The microorganisms are evolved bysequentially culturing microorganisms in media comprising decreasingconcentrations of acetate. This process preferably involves sequentiallyculturing the microorganisms in aliquots of media, with sequentialaliquots comprising decreasing concentrations of acetate. Theconcentrations of acetate in the media are preferably within a range offrom about 0 mg/L to about 80 g/L, such as from about 0.001 mg/L toabout 80 g/L, about 0.01 mg/L to about 50 g/L, about 0.1 mg/L to about10 g/L, or about 0.1 mg/L to about 3 g/L. In some versions, the startingacetate concentration in the medium is within a range of from about 90mg/L to about 80 g/L and sequentially reduces to a concentration with arange of from about 0 mg/L to about 90 mg/L. In some versions, thestarting acetate concentration in the medium is within a range of fromabout 90 mg/L to about 80 g/L and sequentially reduces to aconcentration with a range of from about 0.001 mg/L to about 90 mg/L. Insome versions, the starting acetate concentration in the medium iswithin a range of from about 90 mg/L to about 1 g/L and sequentiallyreduces to a concentration with a range of from about 0.1 mg/L to about90 mg/L. In some versions, the starting acetate concentration in themedium is within a range of from about 90 mg/L to about 500 g/L andsequentially reduces to a concentration with a range of from about 1mg/L to about 90 mg/L.

The initial amount of total consumable carbon in the various media usedin the sequential culturing is preferably approximately the same amongthe media. The initial amount of total consumable carbon preferablyranges from about 1 g/L to about 100 g/L, but may be higher or lower.Beyond the acetate, the balance of consumable carbon preferablycomprises a sugar such as glucose or other carbohydrates or carbonsources known in the art. The sequential culturing may comprise passingthe microorganism through the media in at least about 2, 3, 4, 5, 7, 10,15, or 20 passages and/or up to about 5, 10, 15, 20, 30, 50 or morepassages.

Some versions of the invention comprise microorganisms configured forincreased production of ethanol. These microorganisms have the enzymesdescribed above for producing pyruvate functionally deleted butadditionally have pyruvate formate lyase functionally deleted.

Pyruvate formate lyases catalyze the reversible conversion of pyruvateand coenzyme-A into formate and acetyl-CoA. Pyruvate formate lyasesinclude enzymes classified under EC 2.3.1.54. An exemplary pyruvateformate lyase is the PFL of E. coli (SEQ ID NO:42), which is encoded bypflB (SEQ ID NO:41). Other pyruvate formate lyases include homologs ofthe E. coli PFL.

In some versions of the invention, a pyruvate formate lyase activatingenzyme in the recombinant microorganism is functionally deleted.Pyruvate formate lyase activating enzymes include enzymes classifiedunder EC 1.97.1.4. Pyruvate formate lyase activating enzymes activatepyruvate formate lyases. Functionally deleting a pyruvate formate lyaseactivating enzyme constitutes a way to functionally delete a pyruvateformate lyase. An exemplary pyruvate formate lyase activating enzyme isthe PFL activase of E. coli (SEQ ID NO:44), which is encoded by pflA(SEQ ID NO:43). Other pyruvate formate lyase activating enzymes includehomologs of the E. coli PFL activase.

The enzymes described herein can be functionally deleted by mutating ordisrupting expression of any one or all of the genes encoding the enzymeor its substituent subunits. Accordingly, the pyruvate dehydrogenase canbe functionally deleted by mutating or disrupting expression of any oneor more of aceE, aceF, and lpdA or homologs thereof. The 2-oxoglutaratedehydrogenase can be functionally deleted by mutating or disruptingexpression of any one or more of lpdA, sucA, and sucB or homologsthereof. The phosphate acetyltransferase can be functionally deleted bymutating or disrupting expression of pta or homologs thereof. Theacetate kinase can be functionally deleted by mutating or disruptingexpression of ackA or homologs thereof. The pyruvate oxidase can befunctionally deleted by mutating or disrupting expression of poxB orhomologs thereof. The lactate dehydrogenase can be functionally deletedby mutating or disrupting expression of ldhA or homologs thereof. Thecytochrome oxidase can be functionally deleted by mutating or disruptingexpression of any one or more of cyoA, cyoB, cyoC, cyoD and cyoE orhomologs thereof. The succinate dehydrogenase can be functionallydeleted by mutating or disrupting expression of any one or more of sdhA,sdhB, sdhC, and sdhD or homologs thereof. The 6-phosphogluconatedehydrogenase can be functionally deleted by mutating or disruptingexpression of gnd or homologs thereof. The glutamate dehydrogenase canbe functionally deleted by mutating or disrupting expression of gdhA orhomologs thereof. The pyruvate formate lyase can be functionally deletedby mutating or disrupting expression of pflB and pflA or homologsthereof.

The microorganisms of the invention may also be modified to increaseexpression of one or more enzymes. Modifying the microorganism toincrease expression of an enzyme can be performed using any methodscurrently known in the art or discovered in the future. Examples includegenetically modifying the microorganism and culturing the microorganismin the presence of factors that increase expression of the enzyme.Suitable methods for genetic modification include but are not limited toplacing the coding sequence under the control of a more active promoter,increasing the copy number of the gene, introducing a translationalenhancer on the gene (see, e.g., Olins et al. Journal of BiologicalChemistry, 1989, 264(29):16973-16976), and/or increasing expression oftransactivators. Increasing the copy number of the gene can be performedby introducing additional copies of the gene to the microorganism, i.e.,by incorporating one or more exogenous copies of the native gene or aheterologous homolog thereof into the microbial genome, by introducingsuch copies to the microorganism on a plasmid or other vector, or byother means. “Exogenous” used in reference to a genetic element meansthe genetic element is introduced to a microorganism by geneticmodification. “Heterologous” used in reference to a genetic elementmeans that the genetic element is derived from a different species. Apromoter that controls a particular coding sequence is herein describedas being “operationally connected” to the coding sequence.

The microorganisms of the invention may include at least one recombinantnucleic acid configured to express or overexpress a particular enzyme.“Recombinant” as used herein with reference to a nucleic acid moleculeor polypeptide is one that has a sequence that is not naturallyoccurring, has a sequence that is made by an artificial combination oftwo otherwise separated segments of sequence, or both. This artificialcombination can be achieved, for example, by chemical synthesis or bythe artificial manipulation of isolated segments of nucleic acidmolecules or polypeptides, such as genetic engineering techniques.“Recombinant” is also used to describe nucleic acid molecules that havebeen artificially modified but contain the same regulatory sequences andcoding regions that are found in the organism from which the nucleicacid was isolated. A recombinant cell or microorganism is one thatcontains a recombinant nucleic acid molecule or polypeptide.“Overexpress” as used herein means that a particular gene product isproduced at a higher level in one cell, such as a recombinant cell, thanin a corresponding cell. For example, a microorganism that includes arecombinant nucleic acid configured to overexpress an enzyme producesthe enzyme at a greater amount than a microorganism that does notinclude the recombinant nucleic acid.

Exogenous, heterologous nucleic acids encoding enzymes to be expressedin the microorganism are preferably codon-optimized for the particularmicroorganism in which they are introduced. Codon optimization can beperformed for any nucleic acid by a number of programs, including“GENEGPS”-brand expression optimization algorithm by DNA 2.0 (MenloPark, Calif.), “GENEOPTIMIZER”-brand gene optimization software by LifeTechnologies (Grand Island, N.Y.), and “OPTIMUMGENE”-brand gene designsystem by GenScript (Piscataway, N.J.). Other codon optimizationprograms or services are well known and commercially available.

Microorganisms of the invention configured to increase production ofethanol may be modified to increase expression of pyruvate decarboxylaseand alcohol dehydrogenase.

Pyruvate decarboxylases catalyze the decarboxylation of pyruvic acid toacetaldehyde and carbon dioxide. Pyruvate decarboxylases include enzymesclassified under EC 4.1.1.1. An exemplary pyruvate decarboxylase is thePDC of Zymomonas mobilis (SEQ ID NO:46), which is encoded by pdc (SEQ IDNO:45). Other pyruvate decarboxylases include homologs of the Z. mobilisPDC.

Alcohol dehydrogenases catalyze the interconversion between alcohols andaldehydes or ketones with the reduction of nicotinamide adeninedinucleotide (NAD⁺ to NADH). Alcohol dehydrogenases include enzymesclassified under EC 1.1.1.1. An exemplary alcohol dehydrogenase is theADH2 of Zymomonas mobilis (SEQ ID NO:48), which is encoded by adhB (SEQID NO:47). Other alcohol dehydrogenases include homologs of the Z.mobilis ADH2.

Increased expression of the pyruvate decarboxylase and/or the alcoholdehydrogenase can be included in a microorganism comprising a functionaldeletion of any of the genes or gene products, or combinations thereof,described herein.

Isocitrate lyase, encoded by aceA in E. coli or homologs thereof, canalso be functionally deleted in any of the microorganisms describedherein.

Homologs include genes or gene products (including enzymes) that arederived, naturally or artificially, from a common ancestral gene or geneproduct. Homology is generally inferred from sequence similarity betweentwo or more genes or gene products. Homology between genes may beinferred from sequence similarity between the products of the genes. Theprecise percentage of similarity between sequences that is useful inestablishing homology varies with the gene or gene product at issue, butas little as 25% sequence similarity (e.g., identity) over 50, 100, 150or more residues (nucleotides or amino acids) is routinely used toestablish homology (e.g., over the full length of the two sequences tobe compared). Higher levels of sequence similarity (e.g., identity),e.g., 30%, 35% 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 99% or more, can also be used to establish homology.Accordingly, homologs of the coding sequences, genes, or gene productsdescribed herein include coding sequences, genes, or gene products,respectively, having at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity to the codingsequences, genes, or gene products, respectively, described herein. Insome versions, homologs of the genes described herein include genes thathave gene products at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical to the gene productsof the genes described herein. Methods for determining sequencesimilarity percentages (e.g., BLASTP and BLASTN using defaultparameters) are described herein and are generally available. Thehomologous gene products should demonstrate comparable activities and,if an enzyme, participate in the same or analogous pathways. “Orthologs”are genes or coding sequences thereof in different species that evolvedfrom a common ancestral gene by speciation. Normally, orthologs retainthe same or similar function in the course of evolution. As used herein“orthologs” are included in the term “homologs.” Homologs also includeparalogs.

For sequence comparison and homology determination, one sequencetypically acts as a reference sequence to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are input into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. The sequence comparison algorithm then calculates thepercent sequence identity for the test sequence(s) relative to thereference sequence based on the designated program parameters. A typicalreference sequence of the invention is a nucleic acid or amino acidsequence corresponding to coding sequences, genes, or gene productsdescribed herein.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see CurrentProtocols in Molecular Biology, F. M. Ausubel et al., eds., CurrentProtocols, a joint venture between Greene Publishing Associates, Inc.and John Wiley & Sons, Inc., (supplemented through 2008)).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity for purposes of defininghomologs is the BLAST algorithm, which is described in Altschul et al.,J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analysesis publicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al., supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always>0)and N (penalty score for mismatching residues; always<0). For amino acidsequences, a scoring matrix is used to calculate the cumulative score.Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff (1989) Proc. Natl.Acad. Sci. USA 89:10915).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001. The above-describedtechniques are useful in identifying homologous sequences for use in themethods described herein.

The terms “identical” or “percent identity”, in the context of two ormore nucleic acid or polypeptide sequences, refers to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the sequence comparison algorithms described above (or otheralgorithms available to persons of skill) or by visual inspection.

The phrase “substantially identical”, in the context of two nucleicacids or polypeptides refers to two or more sequences or subsequencesthat have at least about 60%, about 65%, about 70%, about 75%, about80%, about 85%, about 90, about 95%, about 98%, or about 99% or morenucleotide or amino acid residue identity, when compared and aligned formaximum correspondence, as measured using a sequence comparisonalgorithm or by visual inspection. Such “substantially identical”sequences are typically considered to be “homologous” without referenceto actual ancestry. Preferably, the “substantial identity” exists over aregion of the sequences that is at least about 50 residues in length,more preferably over a region of at least about 100 residues, and mostpreferably, the sequences are substantially identical over at leastabout 150 residues, at least about 250 residues, or over the full lengthof the two sequences to be compared.

Accordingly, homologs of the genes described herein include genes withgene products at least about 80%, 85%, 90%, 95%, 97%, 98%, 99%, or moreidentical to the gene products of the genes described herein.

The microorganisms of the invention may be prokaryotic, such as bacteriaor archaea, or eukaryotic, such as yeast. Among bacteria, any bacteriumin the domain Bacteria, the kingdom Eubacteria, the phylumProteobacteria, the class Gammaproteobacteria, the orderEnterobacteriales, and the family Enterobacteriaceae are suitable.Gram-positive, gram-negative, and ungrouped bacteria are suitable.Phototrophs, lithotrophs, and organotrophs are also suitable. Inexemplary versions of the invention, the microorganism is E. coli. Insome versions of the invention, the microorganism is a cyanobacterium.Suitable cyanobacteria include those from the genuses Agmenellum,Anabaena, Aphanocapsa, Arthrosprira, Gloeocapsa, Haplosiphon,Mastigocladus, Nostoc, Oscillatoria, Prochlorococcus, Scytonema,Synechococcus, and Synechocystis. Preferred cyanobacteria include thoseselected from the group consisting of Synechococcus spp., spp.,Synechocystis spp., and Nostoc spp.

An aspect of the present invention includes methods of producingcommodity chemicals, such as pyruvate and/or ethanol, with themicroorganisms of the invention. The methods involve culturing themicroorganism in conditions suitable for growth of the microorganism.Such conditions include providing suitable carbon sources for theparticular microorganism along with suitable micronutrients. Foreukaryotic microorganisms and heterotrophic bacteria, suitable carbonsources include various carbohydrates. Such carbohydrates may includebiomass or other suitable carbon sources known in the art. Forphototrophic bacteria, suitable carbon sources include CO₂, which isprovided together with light energy. The commodity chemical can bepurified or isolated with methods known in the art.

In some versions of the invention, the microorganism may be cultured ina medium comprising a biomass hydrolysate. The biomass hydrolysate canbe produced from any biomass feedstock. Exemplary types of biomassfeedstocks include sucrose-rich feedstocks such as suger cane; starchymaterials, such as corn grain; and lignocellulosic biomass, such ascostal Bermuda grass, corn cobs, corn stover, cotton seed hairs,grasses, hardwood stems, leaves, newspaper, nut shells, paper, primarywastewater solids, softwood stems, solid cattle manure, sorted refuse,swine waste, switchgrass, waste papers from chemical pulps, wheat straw,wood, and woody residues.

Prior to hydrolysis, the biomass feedstock may be pretreated ornon-pretreated. Pretreatment of biomass feedstock removes a largeproportion of the lignin and other materials and enhances the porosityof the biomass prior to hydrolysis. The biomass feedstock may bepretreated by any method. Exemplary pretreatments include chipping,grinding, milling, steam pretreatment, ammonia fiber expansion (AFEX,also referred to as ammonia fiber explosion), ammonia recyclepercolation (ARP), CO₂ explosion, steam explosion, ozonolysis, wetoxidation, acid hydrolysis, dilute-acid hydrolysis, alkaline hydrolysis,organosolv, and pulsed electrical field treatment, among others. See,e.g., Kumar, P.; Barrett, D. M.; Delwiche, M. J.; Stroeve, P., Methodsfor Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis andBiofuel Production. Industrial & Engineering Chemistry Research 2009,48, (8), 3713-3729.

The pretreated or non-pretreated biomass may be hydrolyzed by anysuitable method. Hydrolysis converts biomass polymers to fermentablesugars, such as glucose and xylose, and other monomeric or oligomericcomponents. Exemplary hydrolysis methods include enzymatic hydrolysis(e.g., with cellulases or other enzymes) and acid hydrolysis (e.g., withsulfurous, sulfuric, hydrochloric, hydrofluoric, phosphoric, nitric,and/or formic acids), among other methods.

Exemplary biomass hydrolysates include AFEX-pretreated corn stoverhydrolysate (ACSH) (Schwalbach et al. Appl. Environ. Microbiol. 2012,78, (9), 3442-3457) and AFEX-pretreated switchgrass hydrolysate (ASGH).

The medium comprising the biomass hydrolysate may comprise at leastabout 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about60%, about 70%, about 80%, about 90%, about 95%, or about 99% biomasshydrolysate by volume or by mass.

The term “increase,” whether used to refer to an increase in productionof an organic acid, an increase in expression of an enzyme, etc.,generally refers to an increase from a baseline amount, whether thebaseline amount is a positive amount or none at all.

The elements and method steps described herein can be used in anycombination whether explicitly described or not.

The singular forms “a,” “an,” and “the” include plural referents unlessthe content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e.,“references”) cited herein are expressly incorporated by reference tothe same extent as if each individual reference were specifically andindividually indicated as being incorporated by reference. In case ofconflict between the present disclosure and the incorporated references,the present disclosure controls.

It is understood that the invention is not confined to the particularconstruction and arrangement of parts herein illustrated and described,but embraces such modified forms thereof as come within the scope of thefollowing claims.

Examples Overview

Microbes produce a variety of useful chemicals. However, most strainshave not evolved to produce compounds at industrially-relevant levels.Metabolic engineering develops biocatalysts to produce desired chemicalsat high rates, yields, and titers. Strains have been engineered toproduce a broad range of products, including transportation fuels (e.g.ethanol, butanol and biodiesel) [1-5], pharmaceuticals (e.g. alkeloids,polyketides, nonribosomal peptides and isoprenoids) [6-11] and bulk andfine chemicals (e.g. amino acids, organic acids, industrial solvents andpolymer precursors) [12-16]. Metabolic engineering strategies involveincreasing production of pathway precursors, recycling redox carriers,improving flux through biosynthesis pathways, reducing toxicintermediate concentrations, and/or increasing tolerance tointermediates and products. Increasing precursor(s) supply is oftenneeded to generate more of a desired downstream product. For example,strains with elevated malonyl-CoA levels were engineered to producephloroglucinol (a polyketide derived from malonyl-CoA) [17], and strainswith higher oxaloacetate levels produced more succinate, threonine andlysine, which are all derived from oxaloacetate[18].

Pyruvate is a central metabolite and precursor to acetyl-CoA and severalamino acids (including alanine, lysine, valine, isoleucine and leucine).Commodity chemicals (e.g. ethanol, acetic acid, lactic acid and acrylicacid), as well as active pharmaceutical ingredients (e.g. polyketidesand isoprenoids) can also be derived from pyruvate. Pyruvate can beconverted into >60 commercial chemicals within five reaction steps.Furthermore, pyruvate itself can be used as a food additive, weight lossagent, and anti-aging skin treatment. Microbial production of pyruvateis an attractive alternative to current chemical processes, which areexpensive and toxic [21].

Escherichia coli, Corynebacterium glutamicum, and Saccharomycescerevisiae strains have been genetically engineered to producepyruvate[19-24]. However, most strains have low yields and use expensivemedium components. Previous E. coli metabolic engineering strategiesfocused on blocking pyruvate consumption pathways to phosphoenolpyruvate(PEP), acetyl-CoA, ethanol, acetate, lactate and formate. Otherstrategies prevented conversion of PEP to oxaloacetate by deleting PEPsynthase, increasing glycolytic flux by deleting F1-ATPase deletionmutant or reducing NADH availability [19-21], and reducing TCA cyclefluxes by deleting α-ketoglutarate dehydrogenase [21]. The highestreported yield is 0.75 g pyruvate/g glucose (78% of the theoreticalmaximum yield) using a thiamin supplemented salts minimal medium.Pyruvate overproducing strains have been further altered to produceother chemicals, including alanine and diacetyl [25].

The present examples design and construct pyruvate strains using agenome-scale metabolic model of E. coli. OptORF [26] was used to searchfor gene deletions that would have high pyruvate yields at their maximalgrowth rate. Four mutant strains were constructed and characterized forgrowth and pyruvate production, and two of the four strains wereadaptively evolved to increase growth rates and further improve pyruvateproduction. The pyruvate strains were further engineered to produceethanol, which is derived from pyruvate. The examples show strainsachieving up to 95% of the maximum theoretic yields for pyruvate. Theexamples also show growth and production of chemicals in bioreactors andwith media containing biomass hydrolysate.

Materials and Methods Strains and Plasmids

E. coli BW25113 and the pCP20 plasmid were obtained from the E. coligenetic stock center (CGSC, Yale University). Single E. coli genedeletion strains were obtained from the Keio collection (OpenBiosystems) and used to construct multiple gene deletion strains (listedin Table 1). To generate mutants with multiple gene deletions, thekanamycin resistance gene (kan) was removed using the pCP20 plasmid[39]. An additional gene was deleted (and kan re-inserted) using P1transduction from a donor Keio mutant and selection on LB agar plateswith 50 μg/mL kanamycin. This process was repeated for each additionalknockout and the gene deletions were verified by PCR. The GLBRCE1strain, pJGG2 plasmid, and its corresponding empty vector (pBBR-DSC5)were obtained from Robert Landick (University of Wisconsin-Madison). ThepJGG2 plasmid is a low copy number plasmid with a lac promoter thatcontrols expression of the Zymomonas mobilis PET cassette genes (pdc andadhB) that encode enzymes to produce ethanol from pyruvate. GLBRCE1lacks ldhA, pflB and ackA and contains pJGG2 and a chromosomal copy ofthe PET cassette inserted in the pflB locus [36].

Media and Culture Conditions

For shake flask and hungate tube experiments, M9 minimal media [44]supplemented with glucose and acetate (at varying concentrations) wasused. Gentamicin was added to the media (at 15 μg/mL) for strainscontaining pJGG2 or pBBR-DSC5 plasmids. All strains were preculturedovernight in Luria Broth (LB), pelleted and washed twice in M9 media,and then resuspended in M9 media with an initial OD600 of 0.01. Foraerobic flask experiments, cultures were grown aerobically in 250 mLflasks containing 100 mL of media.

For anaerobic hungate tube experiments, cultures were grown in hungateculture tubes with 10 mL of media and IPTG was added (at 200 μM) toinduce the expression of PET cassette. Hungate tubes were vacuumed andflushed with argon three times. All experiments were carried out intriplicate at 37° C. in a shaking incubator. Samples were periodicallytaken for further analysis and cells were removed using 0.2 μm nylonfilter.

For aerobic bioreactor experiments, a minimal salts medium (adapted from[40]) was used that included 3.5 g/L KH₂PO₄, 5 g/L K₂HPO₄, 3.5 g/L(NH₄)₂HPO₄, 2 mM MgSO₄, 0.1 mM CaCl₂, 0.01 mM FeCl₃ and 0.5 mL per Ltrace metal solution (described previously [40]). Glucose (30 g/L) andacetate (at reported concentrations) were added to the minimal saltsmedium. AFEX-pretreated switchgrass hydrolysate (ASGH) was provided bythe Great Lakes Bioenergy Research Center. The initial concentrations ofglucose, xylose and acetate in ASGH hydrolysate were quantified by HPLC.Bioreactor seed cultures were prepared by inoculating 100 mL of minimalsalts medium (with 30 g/L glucose and 0.9 g/L acetate) from a 5 mLovernight LB culture such that the initial OD600 was 0.01. Cells weregrown at 37° C. for 14 hours in a 250-mL shake flask and thentransferred into three 250-mL flasks containing 100 mL of same medium.The cultures were grown at 37° C. for another 8 hours and used toinoculate the bioreactors. The starting OD600 in the bioreactors was0.05.

Bioreactors

Batch and fed-batch experiments were conducted in a 3 L bioreactor(Applikon Biotechonology, Inc., Shiedam, Netherlands) using a 1 Lworking volume with the following parameters 37° C., 0.5 L/min airinflow and pH 7.0±0.1. Acid (0.5 M H₂SO₄) and base (2 M KOH) bufferswere added to adjust the pH as needed. The stifling speed was set to500-800 rpm by a single Rushton impeller to ensure the dissolved oxygenlevel was above 40% of saturation. Each bioreactor experiment wasconducted in duplicate. Samples were taken periodically for sugar andend-product analysis after cells were removed by centrifugation. Forfed-batch experiments, a 200 g/L acetate solution was added to thereactor when growth slowed. For PYR020, the fed-batch started with 30g/L glucose and 0.6 g/L acetate, and an additional 0.3 g/L acetate wasadded (1.5 mL of 200 g/L solution). For PYR004, the fed-batch startedwith 30 g/L glucose and 1.5 g/L acetate, and an additional 1.5 g/Lacetate was added (7.5 mL of 200 g/L solution).

Chemical Analyses

Glucose concentrations were determined using an enzyme assay from Sigma(GAGO20). Pyruvate, lactate, acetate, succinate, and formateconcentrations in the medium were measured by HPLC using an AminexHPX-87H with Cation-H guard column (Bio-Rad, cat#125-0140). The mobilephase contained 0.02 N H₂SO₄ (for samples from minimal medium) or 0.05 NH₂SO₄ (for samples from ammonia fiber expansion (AFEX)-pretreatedswitchgrass hydrolysate (ASGH)) and was run at a flow rate of 0.5 mL/minat 50° C. The end-products were quantified (from standard curves) basedon their refractive index. The reported yields were all adjusted bytaking into account evaporation and buffer addition to bioreactors. Theuptake and secretion rates were determined from the metabolite andbiomass concentration data during exponential growth. Biomassconcentrations (gram of cell dry weight per liter, gDW/L) werecalculated from OD600 values using a conversion factor 1 OD600=0.415gDW/L [41].

Adaptive Evolution

PYR001 and PYR002 were adaptively evolved independently for 20 passages.The initial cultures were grown in M9 minimal medium with 1.6 g/Lglucose and 0.4 g/L acetate. At an OD600 ˜0.2, cells were transferred tofresh medium (such that starting OD600 was 0.01). During adaptiveevolution, the amount of acetate in the medium was gradually reduced,while the glucose concentration increased so that the total carbonsource was 2 g/L. After 15 passages, the medium contained 1.98 g/Lglucose and 0.02 g/L acetate. Cultures from each passage were frozen andstored at −80° C.

Strain Design

OptORF was used to identify gene deletions that couple growth andpyruvate production [26]. This method finds mutants that would producepyruvate at their highest biomass yield. OptORF was run using a tiltedinner objective function (growth rate—0.001•pyruvate production rate)[42] and a gene deletion penalty equal to 1 in the outer objectivefunction. All simulations were done for glucose aerobic conditions usingthe iJR904 E. coli genome-scale metabolic network [43], with a maximumglucose uptake rate of 10 mmol/gDW/hour and an unlimited oxygen uptake.

Results In Silico Strain Design for Pyruvate Production

To improve pyruvate production, OptORF suggested four strategies whichdelete: (1) aceE, cyoA, cydB, pta, eutI, ldhA and dld; (2) lpdA, gnd,sdhA, poxB, pflB, pflD, tdcE and purU; (3) aceE, gdhA, poxB, ldhA, dld,atpE, pflB, pflD and tdcE; or (4) aceE, gnd poxB, ldhA, dld, atpE, pflB,pflD and tdcE (FIGS. 2A-2D). Given the large numbers of deletions, theidentified genes were further evaluated and prioritized for deletion.Enzymes that are inactive under glucose aerobic conditions (e.g. due toregulation) were first excluded, including pyruvate formate lyases (PflBand PflD) [27, 28]. In addition, eutI, dld and tdcE encode minorisozymes for Pta, LdhA and PflB, respectively [29-32]. Deleting purUalso had little impact on cell growth in glucose minimal media [33, 34].Based on these considerations, pflB, pflD, eutI, dld, tdcE and purU werenot deleted since they are likely to have low (if any) activity anyway.Additionally, the cydB and atpE deletions were experimentally lethal incombination with other suggested gene deletions (data not shown) andwere not included in the constructed strains. The remaining genesidentified by OptORF were deleted to create four engineered strains(PYR001-PYR004, Table 1).

The engineered strains each involved deletions that impacted metabolismand pyruvate production differently. Deleting aceE, lpdA, pta, poxB,and/or ldhA reduces the conversion of pyruvate into acetyl-CoA, acetate,and lactate. Deletion of cyoA, sdhA, and/or lpdA slows down the citricacid (TCA) cycle which would decrease ATP production, and thus biomassyields. With regard to gdhA and gnd, E. coli has two primary pathwaysfor glutamate synthesis using NADPH, ammonia and α-ketoglutarate. Theglutamate dehydrogenase (GDH) pathway (via gdhA) does not require ATP,while the other glutamine synthetase-glutamine oxoglutarateaminotransferase (GS-GOGAT) pathway consumes one ATP per glutamateformed. Deleting gdhA forces cells to use the GS-GOGAT pathway,increasing ATP consumption and decreasing biomass yields. Similarly,deleting gnd prevents NADPH production via the pentose phosphatepathway, and cells produce NADPH from NADH via pyridine nucleotidetranshydrogenase. The transhydrogenase consumes energy, thereby loweringthe maximum biomass yield. In both cases, lowering the maximum biomassyield (via gdhA or gnd deletions) will increase pyruvate yields, sincepyruvate and biomass formation compete for carbon. The gene deletionseither prevent pyruvate consumption or reduce growth, andsynergistically enhance pyruvate production. Based on the computationalresults, four strains (PYR001-PYR004) were constructed and testedexperimentally (see Table 1). The aceA deletion in PYR001 is notrequired.

TABLE 1 Strains and plasmids. Strains/Plasmid Genotype/Relevantcharacteristics Reference E. coli strains BW25113 lacI^(q) rrnBT14ΔlacZWJ16 hsdR514 [39] ΔaraBADAH33 ΔrhaBADLD78 PYR001 BW25113 aceE::kanΔcyoA Δpta This study ΔldhA ΔaceA PYR002 BW25113 lpdA::kan Δgnd ΔpoxBΔsdhA This study PYR003 BW25113 aceE::kan ΔgdhA ΔpoxB ΔldhA This studyPYR004 BW25113 aceE::kan Δgnd ΔpoxB ΔldhA This study PYR010 Adaptivelyevolved strain of PYR001 This study (single isolate) PYR020 Adaptivelyevolved strain of PYR002 This study (single isolate) GLBRCE1 MG1655ΔackA ΔldhA ΔpflB::PET [36] crl(70insIS1) ylbE(253insG) gltB(G3384A)yodD(A85T) glpR(150delG) gatC(916insCC), pJGG2 EH010-pflB PYR010 ΔaceEpflB::kan pJGG2 This study EH020-pflB PYR020 ΔlpdA pflB::kan pJGG2 Thisstudy EH030-pflB PYR003 ΔaceE pflB::kan pJGG2 This study EH040-pflBPYR004 ΔaceE pflB::kan pJGG2 This study Plasmids pBBR1-MSC5 pBBR oriT;P_(lac); Gent^(R) [36] pJGG2 pBBR1-MSC5 with adhB and pdc (PET [36]cassette) from pLOI295; Gent^(R) Abbreviations: kan, kanamycinresistance gene; Gent^(R), gentamicin resistance.

Characterization of Engineered Pyruvate Strains

Pyruvate production was characterized in the parent E. coli (BW25113)and four mutant strains PYR001, PYR002, PYR003 and PYR004 in M9 minimalmedium supplemented with glucose (FIGS. 3A-3C). All mutant strainscontain either an aceE or lpdA deletion, which prevents synthesis ofacetyl-CoA from pyruvate via pyruvate dehydrogenase. As a result,acetate was added to the media for all four mutant strains to allow foracetyl-CoA synthesis and growth (Table 2). The four mutants grew slowerthan the parent strain, but produced pyruvate as predicted by the model(FIGS. 3A-3C), whereas the parent strain did not secrete any pyruvate.Strain PYR001 grew the slowest and only consumed ˜40% of glucose (˜4.0mM) within 60 hours. However, PYR001 converted most of the glucoseconsumed to pyruvate (79% of the theoretical maximum yield, Table 2).Strains PYR003 and PYR004 both completed growth within 20 hours andproduced 17.0 and 19.4 mM pyruvate, respectively (79% and 87% oftheoretical maximum yield). Among the four mutants, PYR002 had thelowest pyruvate yield (43%) and also exhibited a slower growth rate.

The secretion of metabolic by-products, such as succinate, formate,acetate, lactate and ethanol, was analyzed using HPLC (FIG. 4). Acetatewas the main byproduct of the parent strain (BW25113). PYR001 and PYR002each produced ˜1 to 2 mM acetate (which was surprising since theyrequired exogenous acetate for growth), while PYR003 and PYR004 consumedacetate, presumably for acetyl-CoA production. PYR002 was the onlystrain that produced lactate (˜9.8 mM), which explains its relativelylow pyruvate yield. Succinate, formate, and ethanol were below thelimits of detection by HPLC.

TABLE 2 Production of pyruvate from the parent and mutant strains inshake flasks. Pyruvate Yield Pyruvate Production Rate M9 Medium withGrowth % of max. Conversion^(‡) Pyruvate Specific^(¶) Glucose AcetateRate theoretical (g pyruvate/ Titer Volumetric (mmol/gDW/ Strains (g/L)(g/L) (hour⁻¹) yield^(†) g substrate) (g/L)^(§) (g/L/hour) hour) BW251132 0 0.59 ± 0.01 0 0 0 0 0 PYR001 1.9 0.1 0.02 ± 0.00 79.15 ± 4.63 0.78 ±0.05 0.62 ± 0.04 0.01 ± 0.00  6.04 ± 0.24 PYR002 1.8 0.2* 0.12 ± 0.0143.24 ± 2.89 0.43 ± 0.03 0.91 ± 0.06 0.02 ± 0.00  5.47 ± 0.04 PYR003 1.90.1 0.45 ± 0.03 79.05 ± 0.63 0.75 ± 0.00 1.50 ± 0.01 0.08 ± 0.00 20.36 ±0.47 PYR004 1.9 0.1 0.30 ± 0.00 86.60 ± 4.12 0.82 ± 0.04 1.71 ± 0.080.07 ± 0.01 19.11 ± 0.25 PYR010 1.98 0.02 0.20 ± 0.04 68.33 ± 7.81 0.67± 0.08 1.39 ± 0.16 0.06 ± 0.00 14.91 ± 1.68 PYR020 1.98 0.02 0.34 ± 0.0095.23 ± 3.12 0.92 ± 0.03 1.95 ± 0.06 0.05 ± 0.00 23.73 ± 0.88 *PYR002required more acetate than other strains to start growth within 48 hour.^(†)Percent of theoretical yield is calculated as the pyruvateconcentration divided by the theoretical maximum production of pyruvate(2 mmol of pyruvate per mmol of glucose). Acetate was also taken accountfor calculating the theoretical maximum production (0.5 mmol of pyruvateper mmol of acetate). The yield was adjusted by the culture volume lossdue to the liquid evaporation in shake flasks under aerobic conditions.^(‡)Conversion is expressed as the gram of pyruvate produced per gram oftotal carbon source (including glucose and acetate). It was adjusted bythe culture volume loss due to the liquid evaporation in shake flasksunder aerobic conditions. ^(§)The reported titer is the concentrationdetermined by HPLC (and does not account for evaporative loss). ^(¶)Thespecific production rate is the pyruvate production rate per gram ofcell dry weight (gDW) during exponential growth. The numbers that followthe ± sign are standard deviations (SD) from triplicate experiments.

Adaptive Evolution to Improve Pyruvate Productivity

Strains PYR003 and PYR004 showed high pyruvate productivity, whilestrains PYR001 and PYR002 exhibited low pyruvate yields and/orproduction rates. All four pyruvate producing strains were designed suchthat at their maximum growth rate pyruvate production would be high.Therefore, an adaptive evolution approach was used to evolve PYR001 andPYR002 and select for faster growth, which should also select for higherpyruvate rates. Adaptive evolution was conducted under aerobicconditions for 20 passages at 37° C. in glucose+acetate M9 minimalmedium. Acetate was added to the medium to enable cell growth, but theconcentration was reduced over adaptive evolution (Table 2). Singlecolonies of the evolved populations, containing progenies of PYR001 andPYR002, were isolated from the last passage and are referred to asPYR010 and PYR020, respectively. The evolved isolates' growth andpyruvate production were characterized (Table 2 and FIGS. 3D-F). Theevolved strains had a 10-fold (PYR010) and 3-fold (PYR020) increase ingrowth rate and ˜2-fold increase in pyruvate titers (PYR010 and PYR020).In terms of pyruvate yield, PYR010 had a 10% lower yield than itsunevolved strain (PYR001) while PYR020 had ˜2-fold increase (PYR020).Interestingly, both evolved strains needed less acetate (5-fold and10-fold decrease) in the medium to support their growth. Among the fourunevolved strains and two evolved strains, PYR020 performed best withrespect to yield and titer, followed by PYR004. Both strains wereselected for further characterization in bioreactors (Table 3).

Culture in High Concentration of Carbon Source and LignocellulosicBiomass

Strains with high yields, titers and volumetric production rates aredesired for industrial application. While our engineered strainsachieved high yields in shake flasks, their titers and volumetricproduction rate were low due to the low glucose concentrations in themedium. Therefore, a minimal salts medium with higher glucoseconcentrations (30 g/L) was used to evaluate production by two of thehigher yielding pyruvate strains (PYR020 and PYR004). Acetate was thelimiting nutrient for both mutants, and thus two differentconcentrations were used in different experiments (0.9 g/L and 1.5 g/Lfor PYR020, and 1.5 g/L and 3 g/L for PYR004). Experiments wereconducted in 1 L volume, pH-controlled bioreactors, and the dissolvedoxygen level was kept above 40% of saturation to maintain an aerobicenvironment.

PYR020 and PYR004 were first grown in batch bioreactors in minimal saltsmedia with 30 g/L glucose plus acetate. Both PYR004 and PYR020 hadslightly higher growth rates, pyruvate yields and titers in mediacontaining less acetate (1.5 g/L for PYR004 and 0.9 g/L for PYR020)(Table 3). For PYR004, higher acetate concentrations significantlyreduced the time required to complete conversion of glucose to pyruvate(from ˜33 hours to ˜20 hours, FIG. 5). However, at the same acetateconcentration (1.5 g/L) PYR020 was faster than PYR004 (FIG. 5, Panel(A), and FIG. 6, Panel (B)), presumably because PYR020 was evolved togrow at lower acetate concentrations. In batch conditions, both strainsexhibited higher volumetric productivities when grown with higheracetate levels (Table 3). The two strains produce pyruvate at varyingamounts during different stages of batch growth. PYR004 produced a largeamount of pyruvate after growth stopped (˜27% and ˜63% of total pyruvateproduced for 3 and 1.5 g/L acetate, respectively) (FIG. 5), while PYR020produced most of the pyruvate during growth (˜91% and 71% for 1.5 and0.9 g/L acetate, respectively) (FIG. 6). In addition, PYR020 had ˜33%higher specific pyruvate production rates (measured in mmolpyruvate/gDW/h) during exponential growth than PYR004 (Table 3).

Both strains were also grown in fed-batch bioreactors, where additionalacetate was added once growth slowed. Compared to the batch results withthe same total amount of acetate (0.9 g/L for PYR020 and 3 g/L forPYR004), both strains produced less pyruvate (˜1.9 and ˜2.2% loweryields for PYR020 and PYR004, respectively) in fed-batch experiments(Table 3, FIG. 5 and FIG. 6). However, both strains had highervolumetric pyruvate production rates when grown in fed-batch compared tobatch growth with the same total amount of acetate. In both batch andfed-batch operation, tradeoffs appear to exist between volumetricproductivities and pyruvate yields, with PYR004 tending to have highervolumetric productivities and PYR020 tending to have higher yields inthe conditions tested (Table 3).

Since PYR020 had slightly higher pyruvate yields in minimal salts mediathan PYR004, PYR020 was further characterized in media derived fromlignocellosic biomass. AFEX-pretreated switchgrass hydrolysate (ASGH)was used in batch bioreactor experiments, and contained 48 g/L glucoseand 2.6 g/L acetate. The natural presence of acetate in ASGH (and otherplant hydrolysates) meant no acetate supplementation was required.Compared to glucose minimal salts media, PYR020 had a similarexponential growth rate in ASGH (˜0.22 hour⁻¹), but entered into aslower linear growth phase after ˜20 hours (FIG. 7). Growth stopped at−80 hours, after all the glucose and most of the acetate (1.8 g/L) wereutilized. However, xylose, another sugar present in ASGH, was hardlyused. While pyruvate titers (40.7 g/L) and pyruvate yields (85.6%) werestill high, the volumetric production rate was substantially lower inASGH then minimal salts media due to slower growth (Table 3).Hydrolysates derived from lignocellulosic biomass contain microbialinhibitors (e.g., feruloyl amide) [35], whose presence reduces growthand xylose conversion. To further increase pyruvate production fromlignocellulosic biomass, improvements in xylose conversion and inhibitortolerance are likely needed.

TABLE 3 Production of pyruvate from the mutant strains in bioreactors.Pyruvate yield Pyruvate Production Rate Medium^(#) Growth % of max.Conversion^(‡) Pyruvate Specific^(¶) Bioreactor Glucose Acetate Ratetheoretical (g pyruvate/ Titer Volumetric (mmol/gDW/ Strains Mode (g/L)(g/L) (hour⁻¹) yield^(†) g substrate) (g/L)^(§) (g/L/hour) hour) PYR020Batch 30 0.9 0.25 ± 0.02 92.35 ± 0.41 0.89 ± 0.01 27.38 ± 0.16 1.01 ±0.01 20.91 ± 1.60 PYR020 Batch 30 1.5 0.23 ± 0.00 89.95 ± 4.72 0.85 ±0.05 26.85 ± 1.60 1.10 ± 0.07 20.06 ± 2.08 PYR020 Fed-batch 30 0.9 0.27± 0.02 90.61 ± 1.46 0.86 ± 0.02 26.73 ± 0.58 1.14 ± 0.02 24.17 ± 2.05PYR004 Batch 30 1.5 0.56 ± 0.03 91.17 ± 0.02 0.87 ± 0.00 27.35 ± 0.010.88 ± 0.00 15.11 ± 4.61 PYR004 Batch 30 3.0 0.52 ± 0.01 86.63 ± 0.400.80 ± 0.01 26.36 ± 0.41 1.17 ± 0.02 11.45 ± 3.55 PYR004 Fed-batch 303.0 0.53 ± 0.03 84.70 ± 2.70 0.77 ± 0.01 25.32 ± 0.43 1.37 ± 0.02 17.09± 6.71 PYR020 Batch* 48 2.6 0.22 ± 0.02 85.63 ± 3.54 0.82 ± 0.04 40.74 ±2.09 0.51 ± 0.04 26.36 ± 3.10 ^(#)The first six experiments were done ina minimal salts medium (not M9) supplemented with glucose and acetate(see methods for details). In the last experiment, the medium was ASGHhydrolysate which contained 48 g/L glucose, 27 g/L xylose and 2.6 g/Lacetate (as determined by HPLC). ^(†)Percent of theoretical yield iscalculated as the pyruvate concentration divided by the theoreticalmaximum production of pyruvate (2 mmol of pyruvate per mmol of glucose).Acetate was also taken account for calculating the theoretical maximumproduction (0.5 mmol of pyruvate per mmol of acetate). The yield wasadjusted by the culture volume loss due to the liquid evaporation inshake flasks under aerobic conditions. ^(‡)Conversion is expressed asthe gram of pyruvate produced per gram of total carbon source (includingglucose and acetate). It was adjusted to account for the volume of addedbuffer to maintain the bioreactor at pH 7. ^(§)The reported titer is theconcentration determined by HPLC (and does not account for the volume ofadded buffer). ^(¶)The specific production rate is the pyruvateproduction rate per gram of cell dry weight (gDW) during exponentialgrowth. The numbers that follow the ± sign are standard deviations (SD)from duplicate bioreactor experiments.

Production of Ethanol by PYR-Derived Strains

Pyruvate is a precursor to many metabolites, fuels, and chemicals. Totest whether the engineered pyruvate strains could produce otherchemicals, we further engineered the strains to convert pyruvate intoethanol. The pJGG2 plasmid was added which contains the PETcassette—pyruvate decarboxylase (pdc) and alcohol dehydrogenase(adhB)—from Zymomonas mobilis under the control of an IPTG inducible lacpromoter. Ethanol production was measured under anaerobic conditionssince producing ethanol recycles NADH generated by glycolysis. However,under anaerobic conditions pyruvate formate lyase (PflAB) convertspyruvate into acetyl-CoA and formate, and so pflB was additionallydeleted from the pyruvate strains to create four ethanol strains:EH010-pflB, EH020-pflB, EH030-pflB and EH040-pflB.

Anaerobic fermentations in M9 minimal media supplemented with glucose(1.98 g/L) and acetate (0.02 g/L) were carried out in hungate tubes.Three control strains were included: the parent strain (BW25113) withempty vector (pBBR1-MSC5), parent strain with pJGG2 plasmid, and anethanol production strain, GLBRCE1 (which lacks ackA, NW, and ldhA andexpresses the PET cassette from the chromosome and pJGG2 plasmid [36]).In the parent strain, expressing the PET cassette using pJGG2 increasedthe growth rate, ethanol yield (by ˜66%), and ethanol production ratecompared to the empty vector (Table 4). The improved growth and ethanolproduction is likely a result of enhanced NADH recycling. Compared tothe parent strain with pJGG2, all strains engineered to produce ethanol(GLBRCE1, EH010-pflB, EH020-pflB, EH030-pflB and EH040-pflB) had lowergrowth rates (Table 4). Three mutants (EH020-pflB, EH030-pflB andEH040-pflB) had between ˜16% and ˜21% higher ethanol yields compared tothe parent strain with pJGG2, and had similar yields to GLBRCE1 (FIG.8A). Two of these mutants (EH020-pflB and EH040-pflB) had highervolumetric productivity than both GLBRCE1 and the parent strain withpJGG2 (Table 4). Additional fermentations were performed using mediumwith more acetate (0.1 g/L with 1.9 g/L glucose) and/or reduced samplingfrequency, and the ethanol yields and byproduct concentrations did notappear to change when more acetate was supplemented (FIGS. 8A and 8B).

TABLE 4 Production of ethanol from the parent and mutant strains.Ethanol yield Ethanol Production Rate M9 Medium with % of max.Conversion^(‡) Specific^(¶) Growth Rate Glucose Acetate theoretical (gpyruvate/ Ethanol Volumetric (mmol/gDW/ Strains^(§) (hour⁻¹) (g/L) (g/L)yield^(†) g substrate) Titer (g/L) (g/L/hour) hour) BW25113 + 0.28 ±0.00 2 0 38.04 ± 1.70 0.19 ± 0.01 0.39 ± 0.02 0.02 ± 0.00  6.26 ± 0.10pBBR1- MSC5 BW25113 + 0.37 ± 0.02 2 0 63.06 ± 2.59 0.32 ± 0.01 0.64 ±0.03 0.04 ± 0.00 11.71 ± 1.09 pJGG2 GLBRCE1 0.16 ± 0.02 2 0 82.21 ± 0.910.42 ± 0.01 0.83 ± 0.01 0.03 ± 0.00 16.08 ± 0.78 EH010-pflB 0.18 ± 0.011.98 0.02 61.81 ± 6.77 0.31 ± 0.03 0.62 ± 0.07 0.02 ± 0.00 16.61 ± 1.15EH020-pflB 0.25 ± 0.02 1.98 0.02 80.23 ± 4.84 0.41 ± 0.02 0.81 ± 0.050.04 ± 0.00 23.10 ± 1.48 EH030-pflB 0.19 ± 0.05 1.98 0.02 79.47 ± 7.120.40 ± 0.04 0.80 ± 0.07 0.02 ± 0.00 19.29 ± 1.12 EH040-pflB 0.22 ± 0.031.98 0.02 84.59 ± 7.03 0.43 ± 0.04 0.85 ± 0.07 0.04 ± 0.00 22.37 ± 2.28^(§)Strains GLBRCE1, EH010-pflB, EH020-pflB, EH030-pflB, and EH040-pflBall contain pJGG2. ^(†)Percent of theoretical yield is calculated as theethanol concentration divided by the theoretical maximum production ofethanol (2 mmol of ethanol per mmol of glucose). Acetate is also takenaccount for calculating the theoretical maximum production (0.67 mmol ofethanol per mmol of glucose). ^(‡)The conversion is expressed as thegram of ethanol produced per gram of carbon. ^(¶)The specific productionrate is the pyruvate production rate per gram of cell dry weight (gDW)during exponential growth. The numbers that follow the ± sign arestandard deviations (SD) from triplicate experiments.

Discussion

Optimizing production of a specific metabolite usually involvesincreasing synthesis of its precursors. Pyruvate is a starting compoundfor synthesizing a variety of biofuels (e.g., ethanol, 1-butanol andisobutanol) and chemicals. A high-yield pyruvate producing strain hasgreat potential for creating strains to produce valuable chemicals. Inthis study, a genome-scale metabolic model of E. coli and OptORF wereused to identify gene deletion targets to improve pyruvate production.Strains constructed based on the computational predictions produced highlevels of pyruvate and adaptive evolution of two strains increasedpyruvate yields, titers and volumetric production rates. Furtherengineering of these platform pyruvate strains resulted in strains withhigh ethanol production.

All the designed strains over-produced pyruvate. The gene targetsprevented pyruvate consumption by removing competing pathways andreduced growth by eliminating more energetically efficient routes forNADPH and glutamate production. The mutations involved shutting down thepentose phosphate pathway, reducing TCA cycle flux, and lowering biomassproduction (FIGS. 2A-2D). All of the mutants were predicted to haveincreased glycolytic fluxes and coupling between growth and pyruvateproduction. Two of the strains immediately exhibited high pyruvateyields, while two other strains were adaptively evolved to improveproduction rates and/or yields.

All the pyruvate strains have pyruvate dehydrogenase subunits deleted(either aceE or lpdA). The model predicted that other pathways (besidespyruvate-formate lyase) could be used to produce acetyl-CoA. Acetyl-CoAcould be made from acetaldehyde via acetaldehyde dehydrogenase (MhpF),where acetaldehyde is produced by threonine degradation and otherreactions. Acetyl-CoA could also be produced by 2-amino-3-ketobutyrateCoA ligase (Kbl) from threonine degradation. However, all of the mutantswere unable to grow in the absence of acetate, suggesting that theseother pathways are not active at high enough levels. Acetate wasconsumed by all the pyruvate strains, except PYR001, presumably togenerate acetyl-CoA by acetyl-CoA synthetase. The amount of acetateavailable (0.34-3.4 mM) was greater than or close to the amountacetyl-CoA needed for biomass (estimated as the product of the biomassconcentration and acetyl-CoA biomass requirement, which is 3.7 mmolacetyl-CoA per gDW) [37]. In the ethanol production study, the mutantswith increased fluxes of ethanol synthesis were observed to grow faster,which is also probably caused by the generation of acetaldehyde and thenconverted to acetyl-CoA, while another possibility is the balancing ofNADH.

When the resulting pyruvate strains were re-engineered for ethanolproduction, three of the resulting strains achieved high ethanol yields(EH020-pflB, EH030-pflB and EH040-pflB) under anaerobic conditions.Deleting pflB and expressing the PET cassette increased ethanol asexpected, except for EH010-pflB. EH010-pflB (derived from PYR010), hadthe lowest yield of the mutants with pflB deletion and PET addition.Among all the strains tested, EH010-pflB is closest genetically toGLBRCE1. Both EH010-pflB and GLBRCE1 have ldhA, pta and pflB deletions.Even though EH010-pflB has two additional deletions, aceE and cyoA,neither gene would be expected to be expressed anaerobically [38]. Thus,the significantly lower ethanol yield in EH010-pflB compared withGLBRCE1 was unexpected. GLBRCE1 was derived from a closely-relatedbackground strain (MG1655, compared to BW25113) and has an extrachromosomal copy of the PET cassette. This additional copy of the PETcassette could lead to higher PET expression levels and ethanolproduction in GLBRCE1. When compared to EH010, EH010-pflB should havereduced formate production (which it does, see FIG. 8A) and increasedavailability of pyruvate. However, EH010-pflB and EH010 exhibitedsimilar ethanol yields (FIG. 8A). For the EH010-pflB strain, only 80% ofthe carbon was recovered in the biomass and measured products (which islower than the other strains) and so it is possible that some othermetabolite (not detected by HPLC) was secreted by EH010-pflB.

Yeast and bacterial strains have previously been engineered for pyruvateproduction [20, 22-24]. The strains usually require additional nutrientsbesides glucose (e.g., yeast extract, tryptone, thiamine) which willincrease the cost for commercial production. An E. coli strain TC44 waspreviously reported to show the highest pyruvate production with 78% oftheoretical yield and 1.2 g/L/hour production rate, when supplementedwith thiamine. Our strain, PYR020, uses only mineral salt medium andreaches significantly higher yield (92% of theoretical yield) and a highproduction rate of 1.01 g/L/hour. This strain also could utilize cheaperhydrolysate feedstock to produce pyruvate with a high yield and titer.While PYR020 requires acetate for growth, acetate is commonly found inlignocellulosic hydrolysates. The PYR020 and PYR004 strains have thehighest pyruvate production yield reported so far, and will be an idealplatform to create new strains to produce other important chemicalsderived from pyruvate.

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We claim:
 1. A microorganism comprising modifications that reduce orablate activity of: one or more enzymes in a first set selected from thegroup consisting of pyruvate dehydrogenase and 2-oxoglutaratedehydrogenase; one or more enzymes in a second set selected from thegroup consisting of phosphate acetyltransferase, acetate kinase, andpyruvate oxidase; and enzymes in a third set comprising: lactatedehydrogenase and one or more enzymes selected from the group consistingof cytochrome terminal oxidase and succinate dehydrogenase; lactatedehydrogenase and one or more enzymes selected from the group consistingof 6-phosphogluconate dehydrogenase and glutamate dehydrogenase; one ormore enzymes selected from the group consisting of cytochrome terminaloxidase and succinate dehydrogenase and one or more enzymes selectedfrom the group consisting of 6-phosphogluconate dehydrogenase andglutamate dehydrogenase; or lactate dehydrogenase, one or more enzymesselected from the group consisting of cytochrome terminal oxidase andsuccinate dehydrogenase, and one or more enzymes selected from the groupconsisting of 6-phosphogluconate dehydrogenase and glutamatedehydrogenase.
 2. The microorganism of claim 1 wherein the one or moreenzymes in the first set are selected from pyruvate dehydrogenase. 3.The microorganism of claim 1 wherein the one or more enzymes in thesecond set are selected from the group consisting of phosphateacetyltransferase and pyruvate oxidase.
 4. The microorganism of claim 1wherein: the one or more enzymes in the first set are selected frompyruvate dehydrogenase; and the one or more enzymes in the second setare selected from the group consisting of phosphate acetyltransferaseand pyruvate oxidase.
 5. The microorganism of claim 1 wherein theenzymes in the third set comprise: lactate dehydrogenase and cytochrometerminal oxidase; lactate dehydrogenase and one or more enzymes selectedfrom the group consisting of 6-phosphogluconate dehydrogenase andglutamate dehydrogenase; or succinate dehydrogenase and6-phosphogluconate dehydrogenase.
 6. The microorganism of claim 1wherein: the one or more enzymes in the first set are selected frompyruvate dehydrogenase; the one or more enzymes in the second set areselected from phosphate acetyltransferase; and the enzymes in the thirdset comprise: lactate dehydrogenase and one or more enzymes selectedfrom the group consisting of cytochrome terminal oxidase and succinatedehydrogenase; lactate dehydrogenase and one or more enzymes selectedfrom the group consisting of 6-phosphogluconate dehydrogenase andglutamate dehydrogenase.
 7. The microorganism of claim 6 wherein theenzymes in the third set comprise: lactate dehydrogenase and cytochrometerminal oxidase; or lactate dehydrogenase and one or more enzymesselected from the group consisting of 6-phosphogluconate dehydrogenaseand glutamate dehydrogenase.
 8. The microorganism of claim 1 wherein:the one or more enzymes in the first set are selected from pyruvatedehydrogenase; the one or more enzymes in the second set are selectedfrom pyruvate oxidase; and the enzymes in the third set comprise one ormore enzymes selected from the group consisting of cytochrome terminaloxidase and succinate dehydrogenase and one or more enzymes selectedfrom the group consisting of 6-phosphogluconate dehydrogenase andglutamate dehydrogenase.
 11. The microorganism of claim 1 furthercomprising a modification that reduces or ablates activity of an enzymeselected from the group consisting of pyruvate formate lyase andpyruvate formate lyase activating enzyme.
 12. The microorganism of claim1 further comprising a modification that enhances expression of pyruvatedecarboxylase and alcohol dehydrogenase.
 13. The microorganism of claim1 wherein the microorganism is a bacterium or a yeast.
 14. Themicroorganism of claim 1 wherein the microorganism is a bacterium. 15.An evolved microorganism produced by sequentially culturing amicroorganism as recited in claim 1 in media comprising decreasingconcentrations of acetate.
 16. The microorganism of claim 15 wherein theconcentrations of acetate in the media range from about 0.1 mg/L acetateto about 3 g/L acetate.
 17. A method of producing a chemical comprisingculturing a microorganism as recited in claim
 1. 18. The method of claim17 wherein the microorganism further comprises a modification thatreduces or ablates activity of pyruvate formate lyase and a modificationthat enhances expression of pyruvate decarboxylase and alcoholdehydrogenase.
 19. The method of claim 17 wherein the chemical isselected from the group consisting of pyruvate and ethanol.
 20. Themethod of claim 17 wherein the culturing comprises culturing themicroorganism in a medium comprising a biomass hydrolysate.